1. Field of the Invention
The present invention relates to genes and corresponding gene products (DNAs, RNAs and proteins) for use in vaccines and diagnostics specific for intracellular infectious agents, in particular for parasite infection, more in particular for trypanosomatids infection, in particular for Leishmania species infection, more in particular for cutaneous lesions inducing Leishmania, more in particular for Leishmania major.
2. Background Information
Leishmania, a member of the trypanosomatid family, is endemic in the tropical regions of America, Africa and the Indian subcontinent, in the sub-tropics of south-west Asia and in the Mediterranean. Infection with different species of the protozoan Leishmania, which is transmitted by sandflies, manifests itself as either self-healing cutaneous lesions, as neurological and cardiac disorders or as fatal visceral infections. The disease caused by Leishmania is generally called leishmaniasis.
Some forms of the disease are anthroponotic (transmitted only between humans), while others are zoonotic (involving a human reservoir). There are over 20 known species of Leishmania of which a dozen are associated with the various forms of leishmaniasis.
Based on the clinical pattern, the leishmaniasis can be classified into three groups. Cutaneous leishmaniasis is the most prevalent, producing skin ulcers which may take more than a year to heal. Mucocutaneous leishmaniasis initially causes similar lesions which may heal but reappear to cause hideous tissue destruction, primarily the nose and the mouth.
Finally, visceral leishmaniasis is a very severe systemic disease which is nearly always fatal if left untreated.
Since the mid-1980s a dramatic increase in the number of opportunistic Leishmania infections has been reported with the spread of the HIV epidemy to areas traditionally endemic for leishmaniasis. Currently the world wide prevalence of leishmaniasis is 12 million cases (see review Liew and O'Donnell, 1993) and 350 millions live in risk areas. The number of new cases of the disease each year being of the order of 1.5 million, of which 500.000 are visceral leishmaniasis.
Visceral leishmaniasis causes large scale epidemics and the number of cases varies greatly between the years. During 1991 there were large epidemics in India and Sudan. The number of cases in India alone may have been of the order of 250.000. It is estimated that visceral leishmaniasis may have killed 75.000 people in 1991.
The impact of cutaneous leishmaniasis is less dramatic, but it causes severe suffering in endemic areas, not least because of the social and psychological trauma associated with disfigurement. The importance attached to this by people in endemica areas is reflected in the old practice of leishmanization, which involves deliberate and risky infection with Leishmania in order to induce life-long immunity at the price of an ulcer and lesion on less exposed parts of the body.
During their life cycle, Leishmania grow in diverse environmental conditions, and differentiate through distinct developmental stages (see reviews Wilson 1990, Smith et al., 1994). In the sandfly vector, they grow as slender, flagellated infectious promastigotes. When the sandfly takes a blood meal, the parasites are transmitted to the human host where they are phagocytosed by resident skin macrophages. Within the macrophage lysosome, the parasites differentiate into round, non-flagellated amastigotes where they replicate until the macrophage is lysed. They are then released to infect other macrophages, or are taken up by a sandfly where they differentiate back to the promastigote stage to repeat their life cycle.
Life long protection achieved by natural or deliberate infection with Leishmania suggests that mass vaccination against this disease is feasible. Progress has been made in human clinical trials using whole killed parasites in vaccines against cutaneous leishmaniasis. However, problems have arisen such as the unavailability of reagents or variations in the host response to challenge infection. Furthermore, subcutaneous vaccination with heat-killed, avirulent or radio-attenuated promastigotes has often been ineffective or worse, exacerbated lesions. Thus, to avoid the risk of exacerbating naturally acquired infections by vaccination, it is essential to develop second generation vaccines, e.g. to use immunologically purified antigen(s) or derivative(s).
The clinical manifestations of human leishmaniasis can be mimicked in various inbred mouse strains following infection with L.major. The majority of strains (eg. CBA, C57BL/6) are relatively resistant to infection, developing only small lesions at the site of parasite inoculation, which are self-healing within a few weeks. In contrast, susceptible strains such as BALB/c mice develop severe-cutaneous lesions with no tendency to resolve and eventual fatal visceralization of the infection. In experimental murine leishmaniasis, it is generally accepted that CD4.sup.+ T cells are responsible for resistance to infection with L.major, although CD8.sup.+ lymphocytes have also been shown to play a role in the immunity against this parasite (Muller et al., 1989, Hill et al., 1989). Animal model studies have led to the delineation of two defined CD4.sup.+ T lymphocyte subsets, Th1 and Th2, which may in part determine the outcome of L.major infection. Detailed discussions on the importance of the Th1/Th2 balance and the cytokine network(s) have been reported in recent reviews (Liew and O'Donnell, 1993; Milon et al., 1995). Briefly, the Th1 response is associated with the production of the cytokines IL-2 and .gamma.-IFN, parasite killing, healing and protection, while the Th2 response is associated with the production of IL-4 and/or IL-10, disease progression and susceptibility (Heinzel et al., 1991; Scott, 1989). This pattern of lymphokine production has also been observed in human leishmaniasis (Reed and Scott, 1993). More recently, involvement of IL-12 in the immune response to Leishmania has been reported. IL-12 facilitates the development of a Th1 response by stimulating the production of .gamma.-IFN and down-regulating that of IL-4 (Heinzel et al, 1993; Sypek et al; 1993).
Many studies using Leishmania major promastigotes derived antigens, such as the surface membranes gp63 and lipophosphoglycan (Russell et al, 1988), soluble extract (Scott et al, 1987) or gp63 epitopes (Yang et al, 1991) have shown that protection can be achieved at various levels in the susceptible BALB/c mice. Similarly, for example dp72 and GP46/M2 have been used against Leishmania donovani and Leishmania amazonensis respectively, but only partical protection has been observed (Rachamim and Jaffe, 1993; McMahon-Pratt et al, 1993).